Method for constructing microwave radiation brain injury model of animal based on regulation and construction of neurocircuit of entorhinal cortex-vision cortex V1 region

Abstract

The application discloses a method for constructing a microwave radiation animal brain injury model based on an entorhinal cortex-visual cortex V1 area neural loop regulation, comprising the following steps: setting a microwave radiation condition, and performing microwave radiation treatment on a test animal; comparing whether the activity of the entorhinal cortex-visual cortex V1 area neural loop of the test animal is inhibited before and after the microwave radiation treatment, and if the activity is inhibited, then constructing a microwave radiation animal brain injury model by using the microwave radiation condition. The application discloses a method for constructing a microwave radiation animal brain injury model by characterizing and confirming the activity of the entorhinal cortex-visual cortex V1 area neural loop, and reveals a new mechanism of a microwave radiation biological effect from a neural loop level, so as to provide a new idea for the research and development of microwave radiation brain injury prevention and treatment drug measures and brain function intervention based on electromagnetic regulation.

Description

Technical Field

[0001] This invention relates to the field of medical technology. More specifically, this invention relates to a method for constructing an animal model of microwave radiation-induced brain injury based on the modulation of neural circuits in the entorhinal cortex-visual cortex V1 area. Background Technology

[0002] Microwaves are electromagnetic waves with frequencies between 0.3 GHz and 300 GHz. They have been classified as Group 2B carcinogens by the International Agency for Research on Cancer, and the potential harm of microwave radiation to human health cannot be ignored.

[0003] Existing research indicates that the brain is one of the sensitive target organs for electromagnetic radiation damage. Epidemiological studies have found that long-term exposure to microwave radiation can lead to headaches, insomnia, abnormal brain waves, neurasthenia, memory loss / impairment, poor concentration, and sleep disorders. Animal studies have confirmed that microwave radiation can cause a decline in spatial reference memory, short-term memory, long-term memory, and motor learning memory in animals, accompanied by behavioral abnormalities such as anxiety and excitement. These findings suggest that microwave radiation under certain conditions can induce neurobehavioral changes, leading to emotional and behavioral abnormalities and cognitive impairment.

[0004] The entorhinal cortex, serving as a crucial hub connecting the hippocampus with numerous cortical and subcortical brain regions, plays a vital role in spatial cognition, learning and memory, and emotional regulation. Superficial neurons (layers II and III) of the entorhinal cortex receive input from the visual cortex and various subcortical regions. This information is integrated within the hippocampus and then transmitted via CA1 to the deeper layers (layers V and VI) of the entorhinal cortex, ultimately reaching the visual cortex and certain subcortical regions. Besides its close connection to the hippocampus, the entorhinal cortex also maintains close fiber connections with other brain regions such as the prefrontal cortex, visual cortex, nucleus accumbens, and amygdala, forming a complex neural network that plays a significant regulatory role in neurobehavior. Recent research indicates that the entorhinal-visual cortex circuit can modulate emotional behavior. However, there are few reports on the effects of microwave radiation on neural circuits. In particular, research on the effects of microwave radiation on the entorhinal cortex-visual cortex circuit and its role in neurobehavioral changes is still lacking. There is a lack of good methods for constructing animal models of microwave radiation-induced brain injury based on neural circuit levels, resulting in a lack of relevant preventive and treatment drugs. Therefore, there is an urgent need to design a technical solution that can overcome the above-mentioned deficiencies to a certain extent. Summary of the Invention

[0005] One object of the present invention is to provide a method for constructing a microwave radiation animal brain injury model based on the regulation of neural circuits in the entorhinal cortex-visual cortex V1 area, so as to at least solve the above-mentioned problems.

[0006] To achieve the objectives and other advantages of this invention, a method for constructing a microwave radiation-induced animal brain injury model based on the modulation of neural circuits in the entorhinal cortex-visual cortex V1 area is provided, comprising:

[0007] Microwave radiation conditions were set up, and experimental animals were subjected to microwave radiation treatment.

[0008] Compare whether the activity of the entorhinal cortex-visual cortex V1 area neural circuit in experimental animals is inhibited before and after microwave radiation treatment. If it is inhibited, then construct a microwave radiation animal brain injury model using the microwave radiation conditions.

[0009] Furthermore, anterograde neurotropic virus was injected into the entorhinal cortex of experimental animals, or retrograde neurotropic virus was injected into the V1 region of the visual cortex of experimental animals. Based on the fluorescence signal intensity of the entorhinal cortex and the V1 region of the visual cortex, the amount of nerve fiber projection from the entorhinal cortex to the V1 region of the visual cortex was calculated. If the amount of nerve fiber projection was significantly reduced, it was determined that the activity of the entorhinal cortex-visual cortex V1 region neural circuit was inhibited.

[0010] Furthermore, it also includes: comparing the morphological structure of the visual cortex tissue of experimental animals before and after microwave radiation treatment; if the morphological structure of the visual cortex tissue changes, then constructing a microwave radiation animal brain injury model using the microwave radiation conditions.

[0011] Furthermore, it also includes: comparing the calcium activity of neurons in the V1 region of the visual cortex of experimental animals before and after microwave radiation treatment; if the calcium activity of neurons in the V1 region of the visual cortex is significantly reduced, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0012] Furthermore, it also includes: activating glutamatergic neurons in the entorhinal cortex using chemical genetic techniques, and comparing the calcium activity of GABAergic neurons in the V1 region of the visual cortex of experimental animals after microwave radiation treatment. If activating glutamatergic neurons in the entorhinal cortex can significantly enhance the calcium activity of GABAergic neurons in the V1 region of the visual cortex of experimental animals after microwave radiation, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0013] Furthermore, it also includes:

[0014] Compare the activity of SST-type GABAergic neurons in the V1 region of the visual cortex of experimental animals before and after microwave radiation treatment. If the activity of SST-type GABAergic neurons in the V1 region of the visual cortex is inhibited, then construct a microwave radiation animal brain injury model using the microwave radiation conditions.

[0015] Furthermore, it also includes: comparing the neuronal firing activity in the V1 region of the visual cortex of experimental animals before and after microwave radiation treatment; if the neuronal firing activity in the V1 region of the visual cortex decreases significantly, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0016] Furthermore, it also includes: activating glutamatergic neurons in the entorhinal cortex using chemical genetic techniques, and comparing the firing activity of neurons in the V1 region of the visual cortex of experimental animals after microwave radiation treatment. If activating glutamatergic neurons in the entorhinal cortex can significantly increase the firing activity of neurons in the V1 region of the visual cortex of experimental animals after microwave radiation, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0017] Furthermore, the microwave radiation conditions include at least the center frequency, power density, repetition frequency, pulse width, specific absorptivity, and radiation time.

[0018] The present invention has at least the following beneficial effects:

[0019] This invention is the first to propose using the entorhinal cortex-visual cortex neural circuit as a sensitive target site for microwave radiation. Utilizing cutting-edge neuroscience technologies such as neurotropic virus tracing, genetically encoded calcium imaging, in vivo multichannel electrophysiology, and chemogenetics, it investigates changes in the morphology and structure of the visual cortex, neuronal firing in the V1 region, and calcium activity after microwave radiation. It also studies the influence of entorhinal cortex glutamatergic neuronal activity regulation on V1 region neuronal activity. The aim is to elucidate the effects of microwave radiation on the projection of nerve fibers in the entorhinal cortex-visual cortex and its circuit regulation mechanism, thereby establishing an animal model of microwave radiation-induced brain injury. The goal is to reveal new mechanisms of the biological effects of microwave radiation at the neural circuit level, providing new insights for the development of drugs for the prevention and treatment of microwave radiation-induced brain injury and for electromagnetically modulated interventions of brain function.

[0020] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0021] Figure 1 This is a virus tracing display of the projection results of nerve fibers from the entorhinal cortex to the visual cortex in one embodiment of the present invention;

[0022] A: After injection of anterograde tracer virus rAAV-hSyn-EGFP-WPRE-hGH pA into the entorhinal cortex, green fluorescent protein (EGFP) was expressed; B: After injection of retrograde tracer virus rAAV-hSyn-mCherry-WPRE-hGH pA into the V1 region of the visual cortex, red fluorescent protein (mCherry) was expressed.

[0023] Figure 2This is an embodiment of the invention showing the effect of microwave radiation on the projection of V1 nerve fibers in the entorhinal cortex-visual cortex of mice;

[0024] A: Changes in nerve fiber projection in the entorhinal cortex-visual cortex 7 days after microwave irradiation; B: Quantitative fluorescence analysis of nerve fiber projection in the entorhinal cortex-visual cortex circuit 7 days after microwave irradiation; Con: control group, MW: microwave irradiation group; N=4, **** indicates P<0.0001.

[0025] Figure 3 This is an embodiment of the present invention showing changes in the morphological structure of the visual cortex tissue in mice 7 days after microwave radiation.

[0026] Con: control group; MW: microwave radiation group; Scale bar: 50μm.

[0027] Figure 4 This is an embodiment of the present invention showing changes in the ultrastructure of mouse visual cortex tissue 7 days after microwave radiation.

[0028] Con: control group; MW: microwave radiation group; Scale bar: AB, 2μm; C, 500nm; D, 1μm.

[0029] Figure 5 This is an embodiment of the invention showing the effect of microwave radiation on the calcium activity of neurons in the V1 region of the visual cortex in mice when exploring new objects;

[0030] A: Schematic diagram of a novel object recognition behavioral experiment based on fiber optic recording using genetically encoded calcium imaging technology; B: Quantitative analysis of the area under the line (AUC) of changes in calcium signals in neurons in the V1 region of the visual cortex before and after microwave irradiation when mice explore novel objects; C: Line graphs and heatmaps of changes in calcium signals in neurons in the V1 region of the visual cortex in mice before and after microwave irradiation when exploring novel objects in the control group (Con) and the microwave irradiation group (MW); N=6, *** indicates P<0.001.

[0031] Figure 6 This invention relates to the effect of microwave radiation on the calcium activity of neurons in the V1 region of the visual cortex in mice exploring novel arms of the Y maze.

[0032] A: Schematic diagram of a behavioral experiment combining fiber optic recordings based on genetically encoded calcium imaging technology and novel Y-maze arms; B: Quantitative analysis of the area under the amplitude of change (AUC) of calcium signals in neurons in the V1 region of the visual cortex before and after microwave irradiation when mice explored novel Y-maze arms; C: Line graphs and heatmaps of changes in calcium signals in neurons in the V1 region of the visual cortex in mice before and after microwave irradiation when mice explored novel Y-maze arms; N=6, ** indicates P<0.01.

[0033] Figure 7 This is an embodiment of the invention showing the effect of microwave radiation on the firing activity of neurons in the V1 region of the mouse visual cortex;

[0034] A: Grid map of action potentials of neurons in the V1 region of the mouse visual cortex before and after microwave radiation; B: Effect of microwave radiation on the firing rate of action potentials of neurons in the V1 region of the mouse visual cortex; C: Distribution of γ-oscillation power density of neurons in the V1 region of the mouse visual cortex before and after microwave radiation; D: Effect of microwave radiation on γ-oscillation power density of neurons in the V1 region of the mouse visual cortex; Con: control group, MW: microwave radiation group; N=4, * indicates P<0.05, ** indicates P<0.01.

[0035] Figure 8 This invention relates to an embodiment of the effect of microwave radiation on the firing activity of neurons in the V1 region of the visual cortex of mice before and after activation of glutamatergic neurons in the entorhinal cortex.

[0036] AB: Effects of activation of glutamatergic neurons in the entorhinal cortex of mice before microwave irradiation on action potential firing rate (A) and gamma oscillation power density (B) of neurons in the V1 region of the visual cortex; CD: Effects of activation of glutamatergic neurons in the entorhinal cortex after microwave irradiation on action potential firing rate (C) and gamma oscillation power density (D) of neurons in the V1 region of the visual cortex; Con: control group, MW: microwave irradiation group, Saline: saline control group, CNO: chemogenetic activation group; N=4, * indicates P<0.05, ** indicates P<0.01.

[0037] Figure 9 This invention relates to the effect of activation of glutamatergic neurons in the entorhinal cortex on calcium activity of GABAergic neurons in the V1 region of the visual cortex in mice exploring new objects after microwave radiation.

[0038] A: Schematic diagram of the experimental model of chemogenetic combined with fiber optic recording and new object recognition behavior; B: Quantitative analysis of the area under the line (AUC) of the changes in calcium signals of GABAergic neurons in the V1 region of the visual cortex in mice exploring new objects after microwave radiation; C: Line graphs and heatmaps of changes in calcium signals of GABAergic neurons in the V1 region of the visual cortex in mice in the control group (Con) and microwave radiation group (MW) exploring new objects; Con: control group, MW: microwave radiation group, Saline: saline control group, CNO: chemogenetic activation group; N=6, vs Con-Saline, ** indicates P<0.01; vs MW-Saline, ## indicates P<0.01.

[0039] Figure 10 This invention relates to the effect of activation of glutamatergic neurons in the entorhinal cortex on calcium activity of GABA neurons in the V1 region of the visual cortex in mice exploring novel arms of the Y maze after microwave irradiation.

[0040] A: Schematic diagram of the behavioral experiment of chemogenetic combined with fiber optic recording and Y-maze novel arm; B: Quantitative analysis of the area under the line (AUC) of the changes in calcium signals of GABAergic neurons in the V1 region of the visual cortex in mice exploring novel arms after microwave irradiation; C: Line graph and heatmap of changes in calcium signals of GABAergic neurons in the V1 region of the visual cortex in mice in the control group (Con) and microwave irradiation group (MW) exploring new objects; Con: control group, MW: microwave irradiation group, Saline: saline control group, CNO: chemogenetic activation group; N=6, vs Con-Saline, **** indicates P<0.0001; vs MW-Saline, # indicates P<0.05.

[0041] Figure 11 This is an embodiment of the invention showing the effect of microwave radiation on the activity of SST neurons in the V1 region of the mouse visual cortex;

[0042] A: Schematic diagram of SST (red) and c-fos (green) expression in neurons of the V1 region of the mouse visual cortex after microwave irradiation; B: Quantitative analysis of the effect of microwave irradiation on the activity of SST neurons in the V1 region of the mouse visual cortex; Con: control group, MW: microwave irradiation group; N=4, * indicates P<0.05, scale bar=10μm. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0044] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0045] The present invention provides a method for constructing a microwave radiation-induced animal brain injury model based on the modulation of the entorhinal cortex-visual cortex V1 area neural circuit, comprising:

[0046] S1. Set microwave radiation conditions and subject experimental animals to microwave radiation treatment;

[0047] For example, microwave radiation conditions are set using factors including frequency, power, and time, and experimental animals are subjected to microwave radiation treatment.

[0048] S2. Compare whether the activity of the entorhinal cortex-visual cortex V1 area neural circuit in the experimental animals before and after microwave radiation treatment is inhibited. If it is inhibited, construct a microwave radiation animal brain injury model using the microwave radiation conditions.

[0049] For example, neurotropic virus tracing is used to determine whether the activity of the entorhinal cortex-visual cortex V1 neural circuit is inhibited.

[0050] This embodiment constructs an animal model of microwave radiation-induced brain injury by characterizing and confirming the activity of the entorhinal cortex-visual cortex V1 region neural circuit. It reveals a new mechanism of the biological effects of microwave radiation at the neural circuit level, providing new ideas for the development of drug measures for the prevention and treatment of microwave radiation-induced brain injury and for brain function intervention based on electromagnetic modulation.

[0051] In another embodiment, an anterograde neurotropic virus is injected into the entorhinal cortex of the experimental animal, or a retrograde neurotropic virus is injected into the visual cortex V1 region of the experimental animal. The amount of nerve fiber projection from the entorhinal cortex to the visual cortex V1 region is calculated based on the fluorescence signal intensity of the entorhinal cortex and the visual cortex V1 region. If the amount of nerve fiber projection is significantly reduced, it is determined that the activity of the entorhinal cortex-visual cortex V1 region neural circuit is inhibited.

[0052] For example, anterograde neurotropic virus (rAAV-hSyn-EGFP-WPRE-hGH pA) is injected into the entorhinal cortex of experimental animals, or retrograde neurotropic virus (rAAV-hSyn-EGFP-WPRE-hGH pA) is injected into the V1 region of the visual cortex of experimental animals.

[0053] For example, after the virus is stably expressed and the injection site is confirmed, the experimental animals are subjected to microwave radiation, anesthesia, euthanasia, and brain tissue is dissected. The brain tissue is embedded by OCT and prepared into frozen sections with a thickness of 20 μm. Anti-fluorescence quenching mounting medium containing DAPI is added, and the expression of green fluorescent protein EGFP in the entorhinal cortex and visual cortex V1 region is observed by confocal fluorescence microscopy to calculate the amount of nerve fiber projection from the entorhinal cortex to visual cortex V1 region.

[0054] In another embodiment, the method further includes: comparing the morphological structure of the visual cortex tissue of experimental animals before and after microwave radiation treatment; if the morphological structure of the visual cortex tissue changes, then constructing a microwave radiation animal brain injury model using the microwave radiation conditions.

[0055] For example, the morphological structure of the visual cortex includes the structure of the visual cortex under an optical microscope and the structure under a transmission electron microscope. The structure under an optical microscope is obtained by observing the morphological structure of the visual cortex and performing microscopic imaging after staining brain tissue sections of experimental animals and control experimental animals after microwave irradiation with hematoxylin and eosin. The structure under a transmission electron microscope is obtained by observing the ultrastructure of the visual cortex and performing imaging after staining brain tissue sections of experimental animals and control experimental animals after microwave irradiation with uranium acetate and lead citrate.

[0056] In another embodiment, the method further includes: comparing the calcium activity of neurons in the V1 region of the visual cortex of experimental animals before and after microwave radiation treatment; if the calcium activity of neurons in the V1 region of the visual cortex is significantly reduced, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0057] For example, calcium activity of neurons in the V1 region of the visual cortex was detected using genetically encoded calcium imaging technology.

[0058] In another embodiment, the method further includes: activating glutamatergic neurons in the entorhinal cortex using chemogenetic techniques, and comparing the calcium activity of GABAergic neurons in the V1 region of the visual cortex of experimental animals after microwave radiation treatment. If activating glutamatergic neurons in the entorhinal cortex can significantly enhance the calcium activity of GABAergic neurons in the V1 region of the visual cortex of experimental animals after microwave radiation, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0059] For example, adeno-associated virus (rAAV-CaMKⅡα-hM3D(Gq)-EYFP) containing muscarinic receptor protein (hM3D) and a glutamatergic neuron-specific promoter (CaMKⅡα) was injected into the entorhinal cortex via stereotactic microinjection to specifically label glutamatergic neurons in the entorhinal cortex; AAV-DIO-JGCaMP6s containing the gene-encoded calcium ion indicator GCaMP were injected into the V1 region of the visual cortex and an optical fiber ceramic ferrule was implanted; 0.5 h before the experiment, N-chlorozapine (CNO) 5 mg / kg was injected intraperitoneally to specifically activate glutamatergic neurons in the entorhinal cortex, while the control group received the same volume of physiological saline; electrophysiological signal acquisition was completed within 2 h after drug administration.

[0060] In another embodiment, the method further includes: comparing the activity of SST-type GABAergic neurons in the V1 region of the visual cortex of experimental animals before and after microwave radiation treatment; if the activity of SST-type GABAergic neurons in the V1 region of the visual cortex is inhibited, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0061] For example, immunofluorescence assays were used to detect the activity of SST neurons in the V1 region of the visual cortex.

[0062] In another embodiment, the method further includes: comparing the neuronal firing activity in the V1 region of the visual cortex of experimental animals before and after microwave radiation treatment; if the neuronal firing activity in the V1 region of the visual cortex decreases significantly, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0063] For example, a multichannel electrophysiological system was used to detect the firing activity of neurons in the V1 region of the visual cortex.

[0064] In another embodiment, the method further includes: activating glutamatergic neurons in the entorhinal cortex using chemogenetic techniques, and comparing the firing activity of neurons in the V1 region of the visual cortex of experimental animals after microwave radiation treatment. If activating glutamatergic neurons in the entorhinal cortex can significantly increase the firing activity of neurons in the V1 region of the visual cortex of experimental animals after microwave radiation, then a microwave radiation animal brain injury model is constructed using the microwave radiation conditions.

[0065] For example, adeno-associated virus (rAAV-CaMKⅡα-hM3D(Gq)-EYFP) containing muscarinic receptor protein (hM3D) and glutamatergic neuron-specific promoter (CaMKⅡα) was injected into the entorhinal cortex via stereotactic microinjection to specifically label glutamatergic neurons in the entorhinal cortex; microfilament electrodes were implanted in the V1 region of the visual cortex; 0.5 h before the experiment, N-chlorozapine (CNO) 5 mg / kg was injected intraperitoneally to specifically activate glutamatergic neurons in the entorhinal cortex, while the control group received the same volume of physiological saline; electrophysiological signal acquisition was completed within 2 h after drug administration.

[0066] In another embodiment, the microwave radiation conditions include at least the center frequency, power density, repetition frequency, pulse width, specific absorptivity, and radiation time.

[0067] The following explanation uses mice as experimental animals.

[0068] 1. Materials and Methods

[0069] 1.1 Establishment of an animal model of microwave radiation

[0070] Ten-week-old wild-type C57BL / N6 mice and Vgat-Cre gene-edited mice with specifically labeled gamma-aminobutyric acid (GABA) neurons were selected, weighing 22±2g, with half males and half females, and randomly divided into a control group and a radiation group. Histopathological analysis and fiber optic recording combined with behavioral tests were performed on 6 animals / group, while neuroviral tracing, neuroelectrophysiological, and immunofluorescence detection were performed on 4 animals / group.

[0071] The high-power microwave radiation simulation source built by the Academy of Military Medical Sciences was used, with a center frequency of 9.375 GHz and an average power density of 12 mW / cm². 2 Peak power density 800W / cm³ 2 The repetition frequency was 62 Hz, the pulse width was 250 ns, the specific absorption rate (SAR) was 2.58 W / kg, and the radiation time was 15 min. Mice were placed in transparent perforated plexiglass boxes, and the radiation boxes were placed in a rotatable radiation table in a microwave anechoic chamber. The microwave source radiated uniformly from top to bottom, while the control group received sham radiation under the same conditions.

[0072] 1.2 Stereotactic injection of AAV and implantation of fiber optic and microfilament electrodes

[0073] Mice were anesthetized with isoflurane inhalation. Using a stereotaxic instrument and a microinfusion pump, 150 nmol of adeno-associated virus (AAV) was injected into either the left or right entorhinal cortex (4.5 mm posterior to the anterior fontanelle, 4.0 mm lateral to it, and 1.8 mm subsurfaced to the skull) or the left or right visual cortex V1 area (2.7 mm posterior to the anterior fontanelle, 3.8 mm lateral to it, and 1.2 mm subsurfaced to the skull). The needles were left in place for 10 minutes, and then fiber optic ceramic ferrules were implanted for detecting neuronal calcium activity. For mice undergoing multi-channel electrophysiological testing, microfilament electrodes were implanted only in the visual cortex V1 area. Dental cement was used for sealing. Four weeks post-surgery, after the virus expression stabilized, mice underwent cardiac perfusion fixation, and frozen sections of brain tissue were examined using a fluorescence microscope to confirm the accuracy of the injection site.

[0074] 1.3 Detection of nerve fiber projections in the entorhinal cortex-visual cortex circuit using viral tracing technology

[0075] Using neurotropic virus tracing technology, mice were injected stereotactically into the entorhinal cortex with anterograde AAV (rAAV-hSyn-EGFP-WPRE-hGH pA) or into the V1 region of the visual cortex with retrograde AAV (rAAV-hSyn-EGFP-WPRE-hGH pA) via microinjection. Four weeks after AAV injection, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and their brains were harvested. The brain tissue was rapidly frozen at -80°C for 10 min on a cryostat, then frozen at -20°C for 2 h. After OCT embedding, the brain tissue was frozen sectioned to a thickness of 20 μm. The sections were mounted after the addition of an antifluorescence quencher (containing DAPI) and observed using a laser scanning confocal microscope. Image analysis was performed using ImageJ software.

[0076] 1.4 Histopathological analysis of the visual cortex

[0077] (1) Light microscopic observation of visual cortex tissue: Seven days after microwave radiation, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. After fixation by cardiac perfusion with 10% buffered formalin, the brain tissue was removed. The brain tissue was fixed with 4% paraformaldehyde, dehydrated with graded ethanol, cleared with xylene, impregnated with paraffin, embedded in paraffin and sectioned. The section thickness was 3μm. After staining with hematoxylin-eosin (HE), the morphology and structure of visual cortex tissue were observed using an optical microscope and microscopic imaging was performed.

[0078] (2) Ultrastructural observation of visual cortex tissue: Seven days after microwave irradiation, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital and then euthanized by decapitation. Brain tissue was dissected on ice, and approximately 1 mm of the visual cortex brain region of the mice was harvested. 3 The tissue was fixed with 2.5% glutaraldehyde for 2 h, followed by fixation with 1% osmium tetroxide for 2 h, dehydrated with a gradient of ethanol and acetone, embedded in Epon 812 resin, and after semi-thin sectioning and positioning, ultrathin sections with a thickness of 70 nm were prepared. After double staining with uranium acetate and lead citrate, the ultrastructure of the visual cortex tissue was observed and photographed using a transmission electron microscope (HITACHI H7650, Japan).

[0079] 1.5 Detection of neuronal calcium activity in V1 region of visual cortex based on genetically encoded calcium imaging technology

[0080] Based on genetically encoded calcium imaging technology, an AAV (rAAV-hSyn-jGCaMP7b) containing the genetically encoded calcium ion indicator GCaMP was injected into the V1 region of the visual cortex via stereotactic microinjection. Four weeks after AAV injection, a fiber optic recording system (Nanjing Biotechnology Co., Ltd., China) was used, combined with the novel object recognition and Y-maze behavioral experiment paradigms. Anymaze behavioral video analysis software was used to record the movement trajectories of mice, marking their exploration of novel objects and novel extra-arm behaviors. Changes in neuronal calcium activity in the V1 region of the visual cortex were detected during these marking behaviors. Using Matlab-based data analysis software on the fiber optic recording system, the Z-score algorithm was employed to analyze the amplitude of calcium signal changes and calculate the changes in the area under the calcium signal line (AUC).

[0081] 1.6 Detection of neuronal firing activity in the V1 region of the visual cortex using a multichannel electrophysiological system

[0082] A copper wire mesh was laid at the bottom of the open field chamber. The ground wire of the 64-channel electrophysiology equipment was connected to the copper mesh, and the experimental mice were connected to the electrophysiology equipment through electrode connection wires. After opening the Central software, the settings were configured to eliminate 50Hz power frequency interference. Then, Spike Panel was opened, and the channels to be recorded were selected for property settings.

[0083] The local field potential recording function is set to LP 250Hz, Sampling Rate 2k / s, and Action Potential Spike.

[0084] The recorded data was filtered from 250Hz to 5kHz. The recorded data was then imported into Neuro Explorer software for analysis of the field potential power density and action potential firing rate.

[0085] 1.7 Chemogenetic activation of glutamatergic neurons in the entorhinal cortex combined with electrophysiological detection of changes in the activity of neurons in the visual cortex

[0086] Adeno-associated virus (rAAV-CaMKⅡα-hM3D(Gq)-EYFP) containing muscarinic receptor protein (hM3D) and a glutamatergic neuron-specific promoter (CaMKⅡα) was injected into the entorhinal cortex via stereotactic microinjection to specifically label entorhinal cortex glutamatergic neurons. For detecting visual cortex neuronal firing activity, microfilament electrodes were implanted in the V1 region of the visual cortex. For detecting calcium activity in visual cortex GABAergic neurons, AAV-DIO-JGCaMP6s containing the gene-encoded calcium ion indicator GCaMP were injected into the V1 region of the visual cortex and implanted with fiber optic ceramic ferrules. 0.5 h prior to the experiment, entorhinal cortex glutamatergic neurons were specifically activated by intraperitoneal injection of 5 mg / kg of N-chlorozapine (CNO), while the control group received the same volume of saline. Electrophysiological signal acquisition was completed within 2 h after drug administration.

[0087] 1.8 Immunofluorescence assay to detect SST neuron activity in V1 region of visual cortex

[0088] Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and their brains were harvested for frozen sectioning. Sections were prepared with a thickness of 20 μm, and sections containing the V1 region of the visual cortex were collected. The sections were fixed in 4% paraformaldehyde for 15 min, then washed with phosphate-buffered saline (PBST-0.025%) containing 0.025% Triton X-100. After incubation at room temperature for 10 min with 0.25% Triton X-100, the sections were washed with PBST-0.025%. An appropriate amount of blocking solution (0.225 g glycine + 1 mL goat serum + 0.1 g bovine serum albumin + 0.01 mL Tween 20 + 8 mL PBST-0.025%) was added to the tissue and blocked at room temperature for 2 hours. The fluid on the tissue was removed, and a mixture of 1:50 diluted somatostatin (SST) primary antibody (SC-74556, SANTA CRUZ) and 1:200 diluted c-Fos primary antibody (#2250, CST), containing 1% BSA and diluted with PBST-0.025%, was added. The mixture was incubated overnight at 4°C. After washing with PBST-0.025%, goat anti-mouse IgG (Alexa) diluted 1:1000 was added. 594,ab150116,Abcam) and goat anti-rabbit IgG (Alexa) The secondary antibody mixture (488, ab150077, Abcam) was incubated at room temperature in the dark for 1.5 h. After washing with 0.025% PBST, anti-fluorescence quencher (containing DAPI) was added, and the slides were mounted and observed using a laser scanning confocal microscope. Image analysis was performed using ImageJ software to calculate the number of activated SST neurons.

[0089] 1.9 Statistical Analysis

[0090] Experimental data are expressed as mean ± standard deviation. Paired t-tests, independent samples t-tests, or two-way ANOVA were performed using SPSS 26.0 statistical software. P < 0.05 was considered statistically significant.

[0091] 2. Experimental Results

[0092] 2.1 Effects of microwave radiation on nerve fiber projection in the entorhinal-visual cortex circuit of mice

[0093] Anterograde viral tracing results showed that 4 weeks after injection of rAAV-hSyn-EGFP-WPRE-hGH pA into the entorhinal cortex, green fluorescent protein (EGFP) expression was observed in both the entorhinal cortex and the V1 region of the visual cortex, with strong fluorescence signals, indicating that nerve fibers in the entorhinal cortex can project anterogradely into the V1 region of the visual cortex. (See...) Figure 1 A.

[0094] Retrograde virus tracing results showed that 4 weeks after injection of rAAV-hSyn-mCherry-WPRE-hGH pA into the V1 region of the visual cortex, red fluorescent protein mCherry expression was observed in both the V1 region of the visual cortex and the entorhinal cortex, with strong fluorescence signals. This indirectly verified that nerve fibers from the entorhinal cortex can project to the V1 region of the visual cortex. See Figure 1 B.

[0095] Quantitative fluorescence analysis showed that 7 days after microwave irradiation, the projection of nerve fibers from the entorhinal cortex to the V1 area of ​​the visual cortex was significantly reduced (P<0.0001). Figure 2 B). This suggests that microwave radiation can inhibit the activity of the entorhinal cortex-visual cortex V1 neural circuit.

[0096] 2.2 Effects of microwave radiation on the morphological structure of mouse visual cortex tissue

[0097] Optical microscopy revealed that 7 days after microwave irradiation, some visual cortical neurons underwent degeneration and necrosis, primarily characterized by nuclear pyknosis and deep staining, shrunken cell bodies in pyramidal neurons, and slightly widened perivascular spaces. (See [link to article]) Figure 3 .

[0098] Transmission electron microscopy revealed that 7 days after microwave irradiation, chromatin condensation and aggregation along the nuclear membrane occurred in some visual cortex neurons, resulting in irregular nuclear shapes and apoptosis; rough endoplasmic reticulum degranulation was also observed; glial cells showed edema; mitochondria swelled, cavitated, and their cristae disappeared; synaptic clefts were blurred, and presynaptic vesicles accumulated; perivascular spaces widened. Figure 4 .

[0099] The above results indicate that microwave radiation can cause damage to the morphological structure of the visual cortex.

[0100] 2.3 Effects of microwave radiation on calcium activity of neurons in the V1 region of the mouse visual cortex

[0101] Fiber optic recording combined with new object recognition experimental results Figure 5 AC results showed that after microwave radiation, when mice explored new objects, the calcium activity of neurons in the V1 region of the visual cortex in the radiation group was significantly reduced compared with the control group (P<0.001). Figure 5 C) indicates that reduced calcium activity in neurons in the V1 region of the visual cortex is involved in microwave-induced changes in the recognition and memory abilities of mice.

[0102] Fiber optic recording combined with Y-maze experiment results ( Figure 6 AC results showed that after microwave radiation, when mice entered the new arm of the Y maze, the calcium activity of neurons in the V1 region of the visual cortex in the radiation group was significantly reduced compared with the control group (P<0.01). Figure 6 C) indicates that reduced calcium activity in neurons in the V1 region of the visual cortex is involved in microwave-induced changes in spatial exploration memory in mice.

[0103] The above results indicate that reduced calcium activity in neurons in the V1 region of the visual cortex is involved in changes in cognitive behavior induced by microwave radiation in mice, suggesting that neurons in the V1 region of the visual cortex may be sensitive to microwave radiation and play an important regulatory role in microwave radiation-induced brain injury.

[0104] 2.4 Effects of microwave radiation on neuronal firing activity in the V1 region of the mouse visual cortex

[0105] Multichannel neurophysiological analysis results showed ( Figure 7 In mice with AD (advanced visual acuity), the firing rate of action potentials in the V1 region of the visual cortex was significantly lower in the radiation group than in the control group after microwave radiation (P<0.01). Figure 7 B); Simultaneously, the average power density of γ-oscillations in neurons of the V1 region of the visual cortex in the radiation group mice was significantly lower than that in the control group (P<0.05). Figure 7 D).

[0106] The above results indicate that microwave radiation can lead to a decrease in neuronal firing activity in the V1 region of the mouse visual cortex.

[0107] 2.5 Activation of glutamatergic neurons in the entorhinal cortex enhances neuronal firing activity in the V1 area of ​​the visual cortex after microwave radiation.

[0108] Results of multichannel neuroelectrophysiological analysis ( Figure 8 The results (AD) showed that before microwave radiation, there were no significant differences in the firing rate of action potentials and the γ-oscillation power density of neurons in the V1 region of the visual cortex among the experimental groups of mice. This indicates that under normal conditions, selective activation of glutamatergic neurons in the entorhinal cortex has no significant effect on the firing activity of neurons in the V1 region of the mouse visual cortex (P>0.05). Figure 8 (AB). After microwave radiation, the firing rate of action potentials in the V1 region of the visual cortex of mice in the radiation group (MW) (P<0.05) and the γ-oscillation power density (P<0.01) were significantly lower than those in the control group (Con). Figure 8 In contrast, the firing rate of action potentials in the V1 region of the visual cortex of mice in the radiation-activated group (MW-ON) was significantly higher than that in the radiation group (MW). (P<0.05) Figure 8 CD).

[0109] The above results indicate that activation of glutamatergic neurons in the entorhinal cortex can significantly enhance the firing rate of action potentials and γ oscillations in neurons in the V1 region of the visual cortex of mice after microwave radiation, suggesting that glutamatergic neurons in the entorhinal cortex play an important positive regulatory role in the changes in neuronal firing activity in the V1 region of the visual cortex induced by microwave radiation.

[0110] 2.6 Activation of glutamatergic neurons in the entorhinal cortex enhances calcium activity in GABAergic neurons in the V1 region of the visual cortex after microwave radiation.

[0111] Numerous studies have demonstrated a close relationship between gamma oscillations and GABAergic neurons. Since microwave radiation can cause a decrease in the firing rate of action potentials and gamma oscillations in neurons in the V1 region of the visual cortex, this study further investigated the influence of the regulation of glutamatergic neuron activity in the entorhinal cortex on calcium activity in GABAergic neurons in the V1 region of the visual cortex in order to clarify the target neuron types downstream of the entorhinal cortex-visual cortex circuit.

[0112] Fiber optic recording combined with new object recognition experimental results Figure 9 AC results showed that after microwave radiation, when mice explored new objects, the calcium activity of GABAergic neurons in the V1 region of the visual cortex was significantly reduced in the radiation group (MW-Saline) compared to the control group (Con-Saline) (P<0.01). Figure 9 B). Simultaneously, calcium activity in GABAergic neurons of the V1 region of the visual cortex in mice in the radiation-activated group (MW-CNO) was significantly enhanced compared to the control activated group (Con-CNO) (P<0.01). Figure 9 B), and no other statistical differences were observed. These results indicate that decreased calcium activity in GABAergic neurons in the V1 region of the visual cortex is involved in microwave-induced changes in recognition and memory abilities in mice, and that activation of glutamatergic neurons in the entorhinal cortex can improve the decreased calcium activity in V1 region GABAergic neurons after microwave radiation.

[0113] Fiber optic recording combined with the results of the Y-maze novel arm experiment ( Figure 10 AC results showed that after microwave radiation, when mice explored new heteroarms, the calcium activity of neurons in the visual cortex of the irradiated group (MW-Saline) was significantly reduced compared with that of the control group (Con-Saline) (P<0.0001). Figure 10 B). Simultaneously, calcium activity in GABAergic neurons of the V1 region of the visual cortex in mice in the radiation-activated group (MW-CNO) was significantly enhanced compared to the control activated group (Con-CNO) (P<0.05). Figure 10 B), with no other statistical differences observed. These results further indicate that decreased calcium activity in GABAergic neurons in the V1 region of the visual cortex is involved in microwave-induced changes in spatial exploration ability in mice, and activation of glutamatergic neurons in the entorhinal cortex can improve the decreased calcium activity in GABAergic neurons in the V1 region of the visual cortex after microwave radiation.

[0114] In summary, the decreased calcium activity of GABAergic neurons in the V1 region of the visual cortex is involved in the changes in cognitive function induced by microwave radiation in mice. Selective activation of glutamatergic neurons in the entorhinal cortex can enhance the calcium activity of GABAergic neurons in the V1 region after microwave radiation, suggesting that glutamatergic neurons in the entorhinal cortex play an important positive regulatory role in the changes in calcium activity of GABAergic neurons in the visual cortex induced by microwave radiation.

[0115] 2.7 Effects of microwave radiation on the activity of SST neurons in the V1 region of the mouse visual cortex

[0116] Since activation of glutamatergic neurons in the entorhinal cortex can enhance calcium activity of GABAergic neurons in the V1 region of the visual cortex after microwave irradiation, this study focused on detecting the activity of SST-type GABAergic neurons in the V1 region of the visual cortex after microwave irradiation in order to further clarify the types of GABAergic neurons downstream of the entorhinal cortex-visual cortex circuit.

[0117] Immunofluorescence staining results ( Figure 11 (AB) results showed that 7 days after microwave irradiation, compared with the control group, the proportion of activated SST neurons in the V1 region of the visual cortex of mice in the irradiation group was significantly decreased (P<0.05). Figure 11 B) indicates that microwave radiation can lead to inhibition of SST neuron activity in the V1 region of the visual cortex, suggesting that SST neurons may be microwave radiation-sensitive target neurons downstream of the entorhinal cortex-visual cortex circuit.

[0118] 3. Conclusion

[0119] In summary, we found that 9.375GHz, 12mW / cm 2Microwave radiation to mice with a SAR value of 2.58 W / kg for 15 minutes resulted in reduced nerve fiber projection from the entorhinal cortex to the visual cortex V1 region, as well as pathological changes in the morphology and ultrastructure of the visual cortex. Reduced calcium activity in visual cortex V1 neurons contributed to microwave radiation-induced cognitive and behavioral changes, accompanied by a decrease in action potentials and gamma oscillation power density. Selective activation of glutamatergic neurons in the entorhinal cortex improved the decrease in action potentials and gamma oscillation power density in the visual cortex V1 region caused by microwave radiation, as well as the reduced calcium activity of GABAergic neurons. Furthermore, microwave radiation inhibited the activity of SST-type GABAergic neurons in the visual cortex V1 region. These findings suggest that microwave radiation can inhibit the entorhinal cortex-visual cortex V1 neural circuit, reducing projections from entorhinal cortex glutamatergic neurons to the visual cortex V1 region, thereby weakening the effect on GABAergic neurons. This leads to reduced calcium activity, firing activity, and gamma oscillations in V1 neurons, ultimately resulting in cognitive changes. Among them, glutamatergic neurons in the entorhinal cortex have a positive regulatory effect on the activity of neurons in the V1 region of the visual cortex after microwave radiation, and SST-type GABA neurons may be microwave radiation-sensitive target neurons downstream of the entorhinal cortex-visual cortex circuit.

[0120] In summary, this study provides a microwave radiation-sensitive entorhinal cortex-visual cortex neural circuit. Combined with techniques such as neurotropic virus tracing, genetically encoded calcium imaging, neurophysiological recording, chemogenetic regulation, and immunofluorescence, it can be used to evaluate the neurobiological effects of microwave radiation and to construct animal brain injury models. Therefore, it has important reference value for the prevention and treatment of microwave radiation-induced brain injury and for electromagnetically modulated brain function intervention.

[0121] The number of devices and processing scale described herein are for simplification of the invention. Applications, modifications, and variations of the method for constructing a microwave radiation-induced animal brain injury model based on the modulation of neural circuits in the entorhinal cortex-visual cortex V1 area according to the present invention will be readily apparent to those skilled in the art.

[0122] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for constructing an animal model of microwave radiation-induced brain injury based on the regulation of neural circuits in the entorhinal cortex-visual cortex V1 area, characterized in that, include: Microwave radiation conditions were set, and experimental animals were subjected to microwave radiation treatment. Compare whether the activity of the entorhinal cortex-visual cortex V1 area neural circuit in experimental animals is inhibited before and after microwave radiation treatment. If it is inhibited, then construct a microwave radiation animal brain injury model using the microwave radiation conditions. The activity of the entorhinal-visual cortex V1 neural circuit in the experimental animals before and after microwave radiation treatment was confirmed by the following method. 1) Detect the activity of SST-type GABAergic neurons in the V1 region of the visual cortex. If the activity of SST-type GABAergic neurons in the V1 region of the visual cortex is significantly inhibited, it is determined that the activity of the entorhinal cortex-visual cortex V1 region neural circuit is inhibited. 2) Activate glutamatergic neurons in the entorhinal cortex using chemogenetic techniques, and detect whether the calcium activity and / or neuronal firing activity of GABAergic neurons in the V1 region of the visual cortex are significantly enhanced. If they are significantly enhanced, it is further confirmed that the circuit activity is inhibited.

2. The method for constructing a microwave radiation-induced animal brain injury model based on the modulation of the entorhinal cortex-visual cortex V1 area neural circuit as described in claim 1, characterized in that, Also includes: Compare the morphological structure of the visual cortex tissue in experimental animals before and after microwave radiation treatment. If the morphological structure of the visual cortex tissue changes, then construct a microwave radiation animal brain injury model using the microwave radiation conditions.

3. The method for constructing a microwave radiation-induced animal brain injury model based on the modulation of the entorhinal cortex-visual cortex V1 area neural circuit as described in claim 1, characterized in that, The microwave radiation conditions include at least the center frequency, power density, repetition frequency, pulse width, specific absorptivity, and radiation time.

Images